The long-term goal of the PI's laboratory is to develop an understanding of the genetic basis of Staphylococcus aureus pathogenesis and find ways to overcome its ability to express resistance to antibiotics. The purpose of this study is to determine precise physiological roles of methionine sulfoxide reductase (Msr) enzymes in this organism. Oxidation of protein bound methionine residues leads to loss of biological activity. Msr enzymes reduce methionine sulfoxide (MetO) generated under oxidative stress and restore protein function. S. aureus cells treated with cell wall-active antibiotics produce elevated amounts of Msr enzymes. S. aureus possesses three genes that encode proteins involved in the reduction of the S-epimer of MetO (msrA1, msrA2, and msrA3) and a gene, msrB, that encodes a protein involved in the reduction of the R-epimer of MetO. Why S. aureus possesses multiple proteins with apparently overlapping or redundant functions, and their roles in physiology and virulence are not clear. It is hypothesized that the genes encoding these Msr proteins are expressed differentially under different environmental conditions and that their products protect S. aureus from stress conditions. To test this hypothesis, various reporter and msr mutant strains have been constructed. The reporter S. aureus strains will be used to determine, under appropriate growth conditions, the expression patterns of the genes that are responsible for producing the four Msr proteins. The three unique msr mutants constructed for this study include an msrB mutant (lacks ability to reduce R-MetO);a triple msr mutant (msrA1, msrA2, msrA3;lacks ability to reduce S-MetO);and a quadruple msr mutant (msrA1, msrA2, msrA3, msrB;lacks ability to reduce either R- or S-MetO). These mutants will be used to determine the precise roles of Msr proteins in staphylococcal physiology and virulence by conducting appropriate in vitro and in vivo experiments. This study will provide extensive research training for Truman State University undergraduate and ATSU graduate students. Completion of the study will provide a better understanding of the significance of four Msr proteins in S. aureus and open avenues to control infections caused by this pathogen. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is a significant human pathogen. Antibiotic treated S. aureus cells produce elevated amounts of methionine sulfoxide reductase (Msr) proteins. Determination of the precise physiological roles of the four Msr proteins will help understand stress tolerance in general and antibiotic resistance in particular in S. aureus, and should suggest new therapeutic strategies for the control of staphylococcal infections.